CN111748641A - Detection method for typing serotype O antigen molecules of providencia O19, O20, O28, O30 and the like - Google Patents
Detection method for typing serotype O antigen molecules of providencia O19, O20, O28, O30 and the like Download PDFInfo
- Publication number
- CN111748641A CN111748641A CN202010649557.3A CN202010649557A CN111748641A CN 111748641 A CN111748641 A CN 111748641A CN 202010649557 A CN202010649557 A CN 202010649557A CN 111748641 A CN111748641 A CN 111748641A
- Authority
- CN
- China
- Prior art keywords
- providencia
- probe
- value
- amplification curve
- mgb
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/16—Primer sets for multiplex assays
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention relates to analysis of O antigens of Providencia (Providencia) serotypes O19, O20, O28, O30, O31 and O36 by using a real-time fluorescent TaqMan polymerase chain reaction rapid detection system based on an MGB probe. The invention takes O antigen gene cluster specific genes of providencia as target genes, designs and screens primers and MGB probes for O antigen typing of providencia, establishes a corresponding real-time fluorescence PCR (RT-PCR) detection method, and provides a credible way for O antigen typing of Aeromonas hydrophila by utilizing a molecular biology means. The MGB probe provided by the invention is used for detecting providencia in stool, urinary tract infection, wound, burn and bacteremia specimens, and performing O antigen typing on the providencia, and has the advantages of high sensitivity, high specificity, rapid detection and the like.
Description
Technical Field
The invention relates to a Taqman-MGB technology for typing providencia O19, O20, O28, O30, O31 and O36 serotype strain O antigens in a sample and a preparation method thereof. The invention also relates to a method for detecting by using the MGB probe.
Background
Providencia, 0.6-0.8X 1.5-2.5 μm, in accordance with the general definition of Enterobacteriaceae, facultative anaerobic, gram-negative rectus, moving with a peritrichous flagellum, no colonization. Oxidative deamination of phenylalanine and tryptophan. Separating from diarrhea, stool, urinary tract infection, wound, burn and bacteremia specimen. Providencia is a opportunistic pathogen that can cause diarrhea, parenteral infection, urinary tract infection, and other diseases. Common providencia are alcalilificiens (p.alcalifactens), providencia rettgeri, providencia latrungii (p.rustigiii), providencia stuartii (p.stuartii). Drug-resistant bacteria have shown an increasing trend with the widespread use of antibiotics and immunosuppressants, but at the serum level, identification using wound antisera is limited by variation, availability and specificity, and a more feasible molecular typing scheme is needed. Bacterial surface polysaccharides are known to be specific to different sera, such as O antigen or capsule, and different strains of providencia can be typed and identified based on the diversity of O antigens.
TaqMan-MGB Real-time fluorescent PCR (Real-time fluorescence PCR) technical principle: mixing a Taqman probe marked with fluorescein with template DNA, completing thermal cycle of high-temperature denaturation, low-temperature renaturation and proper-temperature extension, observing the polymerase chain reaction rule, cutting off the Taqman probe complementarily matched with the template DNA, dissociating the fluorescein in a reaction system, emitting fluorescence under the excitation of specific light, increasing the amplified target gene segment in an exponential manner along with the increase of cycle times, and obtaining the Ct value by detecting the fluorescence signal intensity which corresponds to the amplified target gene segment and changes along with the amplification in real time.
The TaqMan fluorescent probe is an oligonucleotide probe, the 5 'end of the TaqMan fluorescent probe carries a fluorescent group, such as FAM, TET, VIC, HEX and the like, and the 3' end of the TaqMan fluorescent probe carries a quenching group, such as TAMRA, BHQ and the like. During PCR amplification, a pair of primers is added, and a specific fluorescent probe is added at the same time, when the probe is complete, a fluorescent signal emitted by a reporter group is absorbed by a quenching group; during PCR amplification, the 5 '-3' exonuclease activity of Taq enzyme cuts and degrades the probe, so that the reporter fluorescent group and the quenching fluorescent group are separated, a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescent molecule is formed when one DNA chain is amplified, and the accumulation of the fluorescence signal and the formation of a PCR product are completely synchronous.
The TaqMan-MGB probe is a new technology improved on the basis of the TaqMan probe in recent years, and MGB molecules are added at the 3' end of the probe, so that the MGB probe can increase the annealing temperature (Tm value) of the probe, and the TaqMan-MGB probe can distinguish the difference of 1 base, is completely paired and has a fluorescent signal; as long as there is a base mismatch, there is no signal. The probe was designed to be 15-30 nt long first, with a Tm of 68-70 ℃. At the 5' end of the probe, the presence of G, which may cause fluorescence quenching, is avoided. The probes were synthesized by AuGCT Biotechnology Corporation (Beijing, China) and have 6-carboxyfluorescein (FAM) and Minor Groove Binding (MGB) moieties at the 5 'and 3' ends, respectively. The length of the primer is about 25 nt, and the Tm is between 55 and 60 ℃. The PCR product produced was between 50-200 bp. Primers were synthesized by Invitrogen (shanghai, china). It may be better if the 3 'end of the forward primer is 1-20 nt (typically 10nt or less) from the 5' end of the probe.
When using Taqman MGB PCR assays, amplification reactions were performed in a total volume of 25. mu.L, including 12.5. mu.L LPremix Ex TaqTM(Takara), 0.3. mu.L of each 10. mu.M of forward and reverse primers, 0.3. mu.L of 10. mu.M probe, 0.2. mu.LROXII, 0.3. mu.L of DNA and 11. mu.l of ddH2And O. One protocol included an initial denaturation step at 95 ℃ for 3 minutes followed by denaturation at 95 ℃ for 15 seconds and annealing at 60 ℃ for 45 seconds for 40 cycles in triplicate in a 7500 real-time PCR system (Applied Biosystems, Foster City, Calif., USA), followed by detection and analysis of results using instrumentation such as ABI 7500. Ct value<30 and the amplification curve is positive.
Disclosure of Invention
To achieve the above object, the present invention discloses an MGB probe for typing providencia O19, O20, O28, O30, O31, O36 serotype O antigen in a sample, the probe containing a 5' reporter dye (FAM) and a 3' non-fluorescent quencher (NFQ), conjugated with Minor Groove Binder (MGB) at the 3' end; the method is mainly characterized in that serotype O antigen typing of providencia O19, O20, O28, O30, O31 and O36 refers to the following steps: the specific gene sequence of O antigen gene cluster of providencia serotype has the nucleotide probe sequence shown in SEQ ID NO 13-SEQ ID NO 18.
The invention relates to a TaqMan primer for typing providencia O19, O20, O28, O30, O31 and O36 serotype O antigens in a sample, which is characterized in that the sequence of the primer is as follows: has the nucleotide sequence shown in SEQ ID NO. 1-SEQ ID NO. 12.
The invention further discloses an MGB probe for typing providencia O antigens O19, O20, O28, O30, O31 and O36 in a sample, and the MGB probe is used for specifically and rapidly detecting the application of the providencia O antigens in typing. Wherein the MGB probe is used for O antigen typing detection in providencia O19, O20, O28, O30, O31 and O36 serotypes. The providencia refers to a crude extract of a pure culture of a sample isolated in any environment suitable for the life of providencia. The experimental result shows that the invention can classify providencia O19, O20, O28, O30, O31 and O36 serotype O antigens at lower DNA concentration.
The invention provides a Taqman-MGB PCR system for detecting serotypes O19, O20, O28, O30, O31 and O36 of providencia in an environment, which comprises the following components: premix Ex TaqTM(Takara), 10. mu.M forward and reverse primers, 10. mu.M probe, ROXII, 0.3. mu.L DNA and ddH2And O. The method is mainly characterized in that the primers and probes designed according to the specific gene sequences of O antigen gene clusters of providencia O19, O20, O28, O30, O31 and O36 serotypes:
(1) the length of a Primer designed by using Primer express3.0 in a specific gene sequence of an O antigen gene cluster of providencia O19, O20, O28, O30, O31 and O36 serotypes is about 25 nt, and Tm is between 55 and 60 ℃; the PCR product is between 50-200 bp. It may be better if the 3 'end of the forward primer is 1-20 nt (typically 10nt or less) from the 5' end of the probe.
(2) The probe designed by the Primer express3.0 in the O antigen gene cluster specific gene sequences of providencia O19, O20, O28, O30, O31 and O36 serotypes has the length of 15-30 nt and the Tm of 68-70 ℃. At the 5' end of the probe, the presence of G, which may cause fluorescence quenching, is avoided, with 6-carboxyfluorescein (FAM) and Minor Groove Binding (MGB) moieties at the 5' and 3' ends, respectively. The invention is the practical application of Taqman-MGB PCR technology, and is used for serotype typing identification of providencia O19, O20, O28, O30, O31 and O36.
According to the technical scheme, the Taqman-MGB PCR technology is introduced into the serotype O antigen typing field of providencia Sv4 for the first time, and the MGB probe for quickly, sensitively, highly accurately and strongly repetitively detecting providencia O19, O20, O28, O30, O31 and O36 serotypes in the sample and the detection method thereof are established.
Drawings
FIG. 1 shows the positive detection of providencia serotype probe; wherein
1-1 is a positive result of O19 serotype probe: o19: ct = 16.93;
1-2 are positive results of O20 serotype probe: o20: ct = 15.92;
1-3 are positive results for O28 serotype probe: o28: ct = 17.91;
1-4 are positive results for O30 serotype probe: o30: ct = 15.7;
1-5 are positive results for O31 serotype probe: o31: ct = 16.85;
1-6 are positive results for O36 serotype probe: o36: ct = 16.5;
FIG. 2 is a specific detection of providencia serotype probe; wherein
2-1 and 2-2 are the detection results of O19 serotype probe specificity: o19-2: ct = 35.93; o19-4: ct = 35.9; o19-5: ct = 32.89; o19-6: ct = 34.92; o19-7: ct = 35.93; o19-8: ct = 34.91; o19-11: ct = 35.93; o19-12: ct = 35.95; o19-13: ct = 34.92; o19-14: ct = 35.94; o19-15: ct = 34.92; o19-16: ct = 33.82; o19-17: ct = 31.81; o19-20: ct = 30.96; o19-21: ct = 32.68; o19-22: ct = 33.23; o19-24: ct = 30.93; o19-25: ct = 33.14; o19-27: ct = 32.76; o19-28: ct = 32.19; o19-29: ct = 31.9; o19-31: ct = 32.92; o19-33: ct = 33.93; o19-36: ct = 32.92; o19-40: no Ct value and no amplification curve; o19-43: ct = 33.94; o19-44: ct = 33.94; o19-45: ct = 33.93; o19-46: ct = 33.93; o19-47: ct = 33.94; o19-48: ct = 33.94; o19-49: ct = 33.93; o19-50: ct = 33.94; o19-51: ct = 33.93; o19-52: ct = 33.93; o19-53: ct = 33.94; o19-54: ct = 33.94; o19-55: ct = 33.95; o19-56: ct = 33.93; o19-57: ct = 33.93; o19-60: ct = 33.92; o19-61: ct = 32.92;
2-3 and 2-4 are detection results of O20 serotype probe specificity: O20O 20-2: ct = 36.99; o20-4: ct = 36.98; o20-5: ct = 35.37; o20-6: no Ct value and no amplification curve; o20-7: ct = 37.09; o20-8: ct = 36.96; o20-11: ct = 36.98; o20-12: ct = 36.95; o20-13: ct = 36.92; o20-14: ct = 37.09; o20-15: ct = 36.94; o20-16: ct = 35.93; o20-17: ct = 37.03; o20-19: ct = 35.38; o20-21: ct = 36.97; o20-22: ct = 36.94; o20-24: ct = 37.06; o20-25: ct = 35.93; o20-27: ct = 35.93; o20-28: ct = 36.93; o20-29: ct = 36.95; o20-31: ct = 32.96; o20-33: ct = 33.79; o20-36: ct = 35.36; o20-40: ct = 33.97; o20-43: ct = 36.93; o20-44: ct = 35.93; o20-45: ct = 36.94; o20-46: ct = 34.95; o20-47: ct = 33.67; o20-48: ct = 36.94; o20-49: ct = 36.95; o20-50: no Ct value and no amplification curve; o20-51: ct = 35.96; o20-52: ct = 34.94; o20-53: ct = 35.85; o20-54: ct = 35.57; o20-55: ct = 36.95; o20-56: ct = 35.95; o20-57: ct = 34.95; o20-60: ct = 36.91; o20-61: ct = 36.96;
2-5 and 2-6 are detection results of O28 serotype probe specificity: o28-2: ct = 37.17; o28-4: ct = 36.93; o28-5: ct = 34.58; o28-6: ct = 36.98; o28-7: no Ct value and no amplification curve; o28-8: ct = 36.95; o28-11: ct = 35.96; o28-12: no Ct value and no amplification curve; o28-13: ct = 36.96; o28-14: no Ct value and no amplification curve; o28-15: no Ct value and no amplification curve; o28-16: no Ct value and no amplification curve; o28-17: ct = 34.78; o28-19: ct = 30.06; o28-20: ct = 33.15; o28-21: ct = 35.32; o28-22: ct = 34.02; o28-24: no Ct value and no amplification curve; o28-25: ct = 35.72; o28-27: ct = 31.5; o28-28: ct = 37.07; o28-29: ct = 33.28; o28-31: ct = 35.37; o28-33: ct = 34.94; o28-36: ct = 32.6; o28-40: ct = 34.45; o28-43: ct = 35.83; o28-44: ct = 32.25; o28-45: no Ct value and no amplification curve; o28-46: ct = 30.83; o28-47: ct = 34.96; o28-48: ct = 36.84; o28-49: ct = 36.67; o28-50: no Ct value and no amplification curve; o28-51: ct = 34.13; o28-52: ct = 34.15; o28-53: ct = 37.21; o28-54: ct = 36.06; o28-55: ct = 37.36; o28-56: no Ct value and no amplification curve; o28-57: no Ct value and no amplification curve; o28-60: ct = 35.53; o28-61: ct = 35.66;
2-7 are the detection results of O30 serotype probe specificity: o30-2: no Ct value and no amplification curve; o30-4: no Ct value and no amplification curve; o30-5: ct = 36.88; o30-6: no Ct value and no amplification curve; o30-7: no Ct value and no amplification curve; o30-8: ct = 37.03; o30-11: ct = 35.04; o30-12: ct = 35.52; o30-13: ct = 37; o30-14: ct = 36.95; o30-15: ct = 37.07; o30-16: ct = 36.99; o30-17: ct = 36.92; o30-19: ct = 35.25; o30-20: ct = 37.03; o30-21: no Ct value and no amplification curve; o30-22: ct = 35.91; o30-24: ct = 35.52; o30-25: ct = 37.18; o30-27: ct = 37.05; o30-28: ct = 37.04; o30-29: no Ct value and no amplification curve; o30-31: ct = 37.39; o30-33: ct = 37.19; o30-36: ct = 37.02; o30-40: ct = 37.04; o30-43: ct = 37; o30-44: ct = 36.98; o30-45: ct = 37.27; o30-46: ct = 37.04; o30-47: no Ct value and no amplification curve; o30-48: no Ct value and no amplification curve; o30-49: no Ct value and no amplification curve; o30-50: ct = 37.17; o30-51: ct = 33.82; o30-52: ct = 35.97; o30-53: ct = 36; o30-54: ct = 34.91; o30-55: ct = 35.94; o30-56: no Ct value and no amplification curve; o30-57: ct = 35.93; o30-60: ct = 32.98; o30-61: ct = 35.82;
2-8 and 2-9 are detection results of O31 serotype probe specificity: o31-2: ct = 36.97; o31-4: ct = 36.92; o31-5: no Ct value and no amplification curve; o31-6: no Ct value and no amplification curve; o31-7: ct = 35.93; o31-8: ct = 35.95; o31-11: ct = 36.94; o31-12: ct = 34.92; o31-13: no Ct value and no amplification curve; o31-14: ct = 35.92; o31-15: ct = 36.93; o31-16: ct = 36.93; o31-17: no Ct value and no amplification curve; o31-19: no Ct value and no amplification curve; o31-20: no Ct value and no amplification curve; o31-21: no Ct value and no amplification curve; o31-22: ct = 37.05; o31-24: no Ct value and no amplification curve; o31-25: ct = 35.94; o31-27: no Ct value and no amplification curve; o31-28: no Ct value and no amplification curve; o31-29: ct = 37.03; o31-31: no Ct value and no amplification curve; o31-33: no Ct value and no amplification curve; o31-36: ct = 35.95; o31-40: no Ct value and no amplification curve; o31-43: no Ct value and no amplification curve; o31-44: ct = 36.97; o31-45: ct = 36.82; o31-46: no Ct value and no amplification curve; o31-47: no Ct value and no amplification curve; o31-48: no Ct value and no amplification curve; o31-49: no Ct value and no amplification curve; o31-50: no Ct value and no amplification curve; o31-51: no Ct value and no amplification curve; o31-52: no Ct value and no amplification curve; o31-53: no Ct value and no amplification curve; o31-54: no Ct value and no amplification curve; o31-55: no Ct value and no amplification curve; o31-56: no Ct value and no amplification curve; o31-57: no Ct value and no amplification curve; o31-60: ct = 36.87; o31-61: no Ct value and no amplification curve;
2-10 and 2-11 are detection results of O36 serotype probe specificity: O36O 36-2: ct = 36.92; o36-4: ct = 34.68; o36-5: ct = 36.82; o36-6: ct = 36.99; o36-7: ct = 35.22; o36-8: ct = 35.26; o36-11: ct = 36.92; o36-12: ct = 35.75; o36-13: ct = 33.89; o36-14: ct = 35.96; o36-15: ct = 34.67; o36-16: ct = 36.94; o36-17: ct = 35.67; o36-19: ct = 33.65; o36-20: ct = 32.02; o36-21: ct = 36.71; o36-22: ct = 33.44; o36-24: no Ct value and no amplification curve; o36-25: ct = 35.92; o36-27: ct = 35.52; o36-28: ct = 31.99; o36-29: ct = 33.81; o36-31: ct = 35.24; o36-33: ct = 36.99; o36-40: ct = 35.9; o36-43: ct = 36.97; o36-44: ct = 35.95; o36-45: ct = 34.91; o36-46: ct = 33.55; o36-47: ct = 36.96; o36-48: ct = 37.09; o36-49: ct = 34.96; o36-50: ct = 36.9; o36-51: ct = 36.92; o36-52: ct = 36.91; o36-53: ct = 37.07; o36-54: ct = 34.43; o36-55: ct = 35.43; o36-56: ct = 36.95; o36-57: ct = 37.08; o36-60: ct = 37.09; o36-61: ct = 36.9.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, the technical means used in the present invention are well known to those skilled in the art. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications in the components and amounts of the materials used in these embodiments can be made without departing from the spirit and scope of the invention. The raw materials and reagents used in the present invention are commercially available.
The providencia strain sources used in the present invention are shown in table 1 below:
TABLE 1 bacterial species used in this experiment
aDepartment of Immunobiology of Bacteria Institute of Microbiology andImmunology University of Lodz←Hungarian National Collection Medical Bacteria(National Insititute of Hygiene,Budapest,Hungary)
Example 1
Design of primers and probes
1. Screening for specific genes
O antigens are synthesized by serotype O antigens of providencia O19, O20, O28, O30, O31 and O36 through two ways, wherein one way is to synthesize the O antigenswzyThe O antigen is synthesized by a dependent pathway, and the O antigen is synthesized by an ABC-transporter pathway. BeforeIn the above-mentioned group, the first group,wzx/wzythe gene is very specific and can be used directly as a candidate for designing a probe, whereas in the latter, there is no image likewzx/wzyThis is a specific gene, so only relatively specific genes can be selected, which will usually be some rare monosaccharide genes or glycosyltransferases. According to the invention, all genes in the gene cluster are compared by the all _ vs _ all _ blast method, and the matching number of specific genes is inevitably far smaller than that of conserved genes. The method is integrated to find out specific genes and design probe primers for the specific genes.
2. Design of primers and probes
The selected specific genes of providencia O19, O20, O28, O30, O31 and O36 serotypes are used as templates to design primers and probes of Taqman-MGB. Processing Using Primer Express3.0wzyThe sequence of the gene was designed as TaqMan MGB probe and primer. The GC% content of the primers and probes is between 40 and 70%, and the formation of a ring hair clamp structure, a self-dimer and a cross-dimer is avoided. The specificity of primers and probes was verified by comparing their sequences to all sequences in GenBank using BLAST search. The number and sequence information for each primer and probe is listed in tables 2 and 3.
The probe was designed to be 15-30 nt long first, with a Tm of 68-70 ℃. At the 5' end of the probe, the presence of G, which may cause fluorescence quenching, is avoided. The probes were synthesized by AuGCT Biotechnology Corporation (Beijing, China) and have 6-carboxyfluorescein (FAM) and Minor Groove Binding (MGB) moieties at the 5 'and 3' ends, respectively.
The length of the primer is about 25 nt, and the Tm is between 55 and 60 ℃. The PCR product produced was between 50-200 bp. Primers were synthesized by Invitrogen (shanghai, china). It may be better if the 3 'end of the forward primer is 1-20 nt (typically 10nt or less) from the 5' end of the probe.
TABLE 2 primers used in Taqman-MGB
TABLE 3 probes used for Taqman-MGB
Example 2
Extraction of sample nucleic acid (isolated from any environment suitable for providencia life, crude extract of pure culture of sample)
1. The sample to be tested is diluted with sterile water, typically at a dilution factor of 1: 10 (e.g. 10g solid sample or 10ml liquid sample in 90ml sterile water) to make a bacterial liquid mother liquor (liquid only).
The bacterial liquid mother liquor is diluted with sterile water for 5 gradients of 1 × 10-1、1×10-2、1×10-3、1×10-4、1×10-5And uniformly coating each gradient bacterial liquid on an LB solid culture medium respectively for culture at 37 ℃. When a single colony grows on the plate, the colony is picked up and inoculated in LB liquid medium and cultured at 37 ℃ overnight at 180rpm (the inoculation operation is carried out in a clean bench).
2. Sample treatment: adding 1 mL of bacterial liquid cultured overnight into a 1.5 mL centrifuge tube, centrifuging at room temperature of 8000 rpm for 1 min, discarding the supernatant, and collecting the thallus. Adding 400 mu L Buffer digest, shaking and mixing uniformly, and carrying out water bath at 65 ℃ for 1 h until the cells are completely cracked.
In the water bath process, the mixture is inverted and uniformly mixed once every 10 min, so that the sample can be promoted to crack, and the mixed solution becomes clear and transparent and is completely cracked;
3. adding 200 mu L Buffer PB, fully reversing and uniformly mixing, and placing in a refrigerator at-20 ℃ for 5 min;
4. centrifuging at room temperature of 10000 rpm for 5 min, and transferring the supernatant (500-550 mu L) into a new 1.5 mL centrifuge tube;
5. adding isopropanol with equal volume, reversing for 5-8 times to mix thoroughly, standing at room temperature for 2-3 min, centrifuging at room temperature 10000 rpm for 5 min, and removing supernatant;
6. adding 1 mL of 75% ethanol, rinsing by inversion for 1-3 min, centrifuging at 10000 rpm for 2 min, and removing the supernatant;
7. repeating the step 6 once;
8. opening the cover and inverting for 5-10 min at room temperature until the residual ethanol is completely volatilized;
9. 50-100 mu L ddH is used for the obtained DNA2Dissolving O, and keeping in a refrigerator at-20 ℃ for later use;
10. and (5) determining the concentration to 300 ng/muL.
Example 3
Positive detection
1. Real-Time PCR System (25 μ L, O19 as an example)
2. Real-Time PCR reaction conditions:
3. the result of the detection
FIG. 1 shows the self-detection results of each serotype probe of providencia, and the results show that: and each reaction system has a good amplification curve, which shows that each serotype detection system can realize accurate detection of the corresponding serotype.
Example 4
Specificity detection
1. Real-Time PCR System (25 μ L, using partial O19 detection as an example)
2. Real-Time PCR reaction conditions:
3. and (3) detection results:
the results show that:
when the genomes of other serotypes were added to the systems of providencia genome samples O19, O20, O28, O30, O31 and O36, the results are shown in fig. 2, and no amplification curve appeared, indicating that the MGB probe specificity of each serotype was good.
Sequence listing
<110> university of southern kayak
<120> detection method for serotype O antigen molecule parting of providencia O19, O20, O28, O30 and the like
<160>18
<170>SIPOSequenceListing 1.0
<210>1
<211>23
<212>DNA
<213> Artificial sequence ()
<400>1
ctcgcatata cgaatgggag tca 23
<210>2
<211>26
<212>DNA
<213> Artificial sequence ()
<400>2
tgtgagtagt gatgtttcaa gcattt 26
<210>3
<211>25
<212>DNA
<213> Artificial sequence ()
<400>3
ttccataaga aaaaagatcg ggaat 25
<210>4
<211>25
<212>DNA
<213> Artificial sequence ()
<400>4
cgaacttatt aggagctgca tacca 25
<210>5
<211>22
<212>DNA
<213> Artificial sequence ()
<400>5
gatggccccc tttgtaattg ta 22
<210>6
<211>23
<212>DNA
<213> Artificial sequence ()
<400>6
cagatgcgcc agataaaatt gag 23
<210>7
<211>26
<212>DNA
<213> Artificial sequence ()
<400>7
cagtatcgtc gctatacagt tcacaa 26
<210>8
<211>25
<212>DNA
<213> Artificial sequence ()
<400>8
accatattca cggtattcag caatg 25
<210>9
<211>22
<212>DNA
<213> Artificial sequence ()
<400>9
gctaggcttt gggtttgatg at 22
<210>10
<211>21
<212>DNA
<213> Artificial sequence ()
<400>10
agccaggttc ccaaccataa g 21
<210>11
<211>24
<212>DNA
<213> Artificial sequence ()
<400>11
acgggtaccc tagattcaca tctt 24
<210>12
<211>23
<212>DNA
<213> Artificial sequence ()
<400>12
cccaagtgat attccaagcg tat 23
<210>13
<211>16
<212>DNA
<213> Artificial sequence ()
<400>13
<210>14
<211>18
<212>DNA
<213> Artificial sequence ()
<400>14
<210>15
<211>20
<212>DNA
<213> Artificial sequence ()
<400>15
<210>16
<211>20
<212>DNA
<213> Artificial sequence ()
<400>16
<210>17
<211>22
<212>DNA
<213> Artificial sequence ()
<400>17
actacactgt aattagtcag ca 22
<210>18
<211>22
<212>DNA
<213> Artificial sequence ()
<400>18
tattcactta ttgactcatt cc 22
Claims (4)
1. A Taqman specific primer for typing providencia O19, O20, O28, O30, O31 and O36 serotype O antigens in a sample is characterized by having a nucleotide sequence shown in SEQ ID NO. 1-SEQ ID NO. 12.
2. An MGB probe comprising a specific primer according to claim 1, the probe comprising a 5' reporter dye (FAM) and a 3' non-fluorescent quencher (NFQ), conjugated at the 3' end to a Minor Groove Binder (MGB); it is mainly characterized by having a nucleotide sequence shown in SEQ ID NO. 13-SEQ ID NO. 18.
3. Use of the Taqman-specific primers and MGB probes for typing O antigens of providencia O19, O20, O28, O30, O31, O36 serotypes in a sample according to claim 1 for the specific rapid detection of O antigen typing of providencia.
4. The use of claim 3, wherein said providencia is selected from the group consisting of: crude extract isolated from pure culture of samples obtained in an environment suitable for providencia.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010649557.3A CN111748641A (en) | 2020-07-08 | 2020-07-08 | Detection method for typing serotype O antigen molecules of providencia O19, O20, O28, O30 and the like |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010649557.3A CN111748641A (en) | 2020-07-08 | 2020-07-08 | Detection method for typing serotype O antigen molecules of providencia O19, O20, O28, O30 and the like |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111748641A true CN111748641A (en) | 2020-10-09 |
Family
ID=72680081
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010649557.3A Pending CN111748641A (en) | 2020-07-08 | 2020-07-08 | Detection method for typing serotype O antigen molecules of providencia O19, O20, O28, O30 and the like |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111748641A (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103993090A (en) * | 2014-05-29 | 2014-08-20 | 南开大学 | Specific nucleotides for providencia O31, O41, O42, O43 and O50 and application of specific nucleotides |
| CN104059975A (en) * | 2014-06-23 | 2014-09-24 | 南开大学 | Providencia O3, O4, O8, O12, O13 and O20 specific nucleotides and application thereof |
| CN110819730A (en) * | 2019-11-25 | 2020-02-21 | 南开大学 | Detection method for typing of Acinetobacter baumannii Sv4 serotype O antigen molecules |
-
2020
- 2020-07-08 CN CN202010649557.3A patent/CN111748641A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103993090A (en) * | 2014-05-29 | 2014-08-20 | 南开大学 | Specific nucleotides for providencia O31, O41, O42, O43 and O50 and application of specific nucleotides |
| CN104059975A (en) * | 2014-06-23 | 2014-09-24 | 南开大学 | Providencia O3, O4, O8, O12, O13 and O20 specific nucleotides and application thereof |
| CN110819730A (en) * | 2019-11-25 | 2020-02-21 | 南开大学 | Detection method for typing of Acinetobacter baumannii Sv4 serotype O antigen molecules |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2021103641A1 (en) | Detection method for molecular typing acinetobacter baumannii sv4 serotype o antigen | |
| WO2022007326A1 (en) | Method and use for real-time fluorescent pcr detection of cronobacter sakazakii o-antigen | |
| Huang et al. | Quadruplex real-time PCR assay for detection and identification of Vibrio cholerae O1 and O139 strains and determination of their toxigenic potential | |
| JP2013500008A (en) | Sequences for the detection and characterization of E. coli O157: H7 and their use | |
| CN110885906A (en) | Compositions and methods for detecting human papillomavirus nucleic acids | |
| CN110699485A (en) | RPA primer pair, probe, kit and detection method for rapidly detecting Marek's disease virus | |
| JP2007274934A (en) | Primer set and method for detecting food poisoning bacterium | |
| WO2015180484A1 (en) | Nucleotides specific to providencia o31, o41, o42, o43 and o50 and uses thereof | |
| CN111748641A (en) | Detection method for typing serotype O antigen molecules of providencia O19, O20, O28, O30 and the like | |
| CN111662993B (en) | Detection method for serotype O antigen serotype molecule typing of vibrio vulnificus Sv4 | |
| CN105256041B (en) | The nucleotide special to aeromonas hydrophila O44, O24, O25 and O28 and application | |
| Amann et al. | Typing in situ with probes | |
| CN112592987A (en) | Real-time fluorescence PCR detection method for 6 serotype vibrio cholerae and application thereof | |
| CN111334613B (en) | RPA primer pair, probe, kit and detection method for detecting canine adenovirus | |
| CN111850144A (en) | Detection method for hydrophilic aeromonas serotype O antigen molecular typing and application | |
| CN112592985B (en) | Real-time fluorescence PCR detection method for bacillus cereus and application | |
| CN111763753A (en) | Detection method for typing of Hafnia alvei PCM1188 serotype O antigen molecules | |
| CN112375834B (en) | PCR detection method and application of 4 main O antigen serotypes of yersinia enterocolitica | |
| CN111850145A (en) | Detection method for serotype O antigen molecule parting of aeromonas hydrophila O7, O16, O19, O24 and the like | |
| CZ298165B6 (en) | Oligonucleotide and method for detecting Pectinatus frisingensis and Pectinatus cerevisiiphilu by making use of such oligonucleotide | |
| CN111621578A (en) | RU 61-00441 gene-based Salmonella spelt constant-temperature detection kit and method | |
| CN111876500A (en) | Detection method for typing of O-type antigen molecules of citric acid bacillus O4 and O24 serotypes | |
| CN111876501A (en) | Real-time fluorescence PCR detection method for enterobacter aerogenes PSgc4 type strain and application | |
| CN114854886A (en) | Real-time fluorescence PCR detection method for Edwardsiella tarda and application thereof | |
| CN109355413A (en) | Staphylococcus aureus fluorescence PCR detection reagent kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20201009 |
|
| WD01 | Invention patent application deemed withdrawn after publication |